Cloning and functional analysis of CmFT in Chinese chestnut

Abstract:【Objective】Flower is the sex organ of plant and the basis for sexual reproduction. Flowering is regulated by external environment and endogenous factors. FLOWERING LOCUS T (FT) is the florigen gene to promote flowering. Under induced condition, FT is expressed in companion cell of leaf sieve tube and transported to the shoot apical meristem activating downstream flowering-related genes. Chinese chestnut (Castanea mollissima) is an important economic forest tree species and mainly distributed in the mountainous regions in China. Nut, the product of sexual reproduction, is the main economic product of Chinese chestnut. However, the flowering of Chinese chestnut is unstable, which usually leads to secondary flowering and“on year and off year”phenomena. The flowering regulatory network in Chinese chestnut is still unclear. This study intends to clone C. mollissima florigen gene CmFT and verify its biological function.【Methods】The TBtools was used to search and obtain CmFT genome sequence information from Chinese chestnut genome database. The CmFT structure diagram was drawn with online software GSDS 2.0. FT amino acid sequences of different species were downloaded from Phytozome database. The phylogenetic tree of FT amino acid sequences in Chinese chestnut and other species was constructed with MEGAX software. The CDD- tools in NCBI was used to search the conserved domains of CmFT. Real-time fluorescence quantitative Polymerase Chain Reaction (PCR) was used to determine the expression characteristics of CmFT in different tissues and leaves at different periods. Vector containing 35S::CmFT-GFP was constructed and transformed in A. thaliana protoplasts to determine the subcellular location information of CmFT by observing the fluorescence signal with laser confocal microscope. 35S::CmFT vector was constructed and transformed in wild type A. thaliana. Five transgenic Arabidopsis lines were selected to verify the biological function of CmFT in flowering by analyzing flowering phenotype. To determine the pathway of CmFT affecting flowering, expressions of endogenous flowering key genes including FT, TERMINAL FLOWER1 (TFL1), CONSTANT (CO), SUPPRESSOR OF CONSTANS OVEREXPRESSION 1 (SOC1), FLOWERING LOCUS C (FLC) and LEAFY (LFY) in transgenic and wild type Arabidopsis plants were determined with real-time fluorescence quantitative PCR.【Results】The FT homologous gene BUA.CMHBY204722 was retrieved in the Chinese chestnut genome database and named CmFT, which contained 3 introns, and the coding sequence (CDS) was 525 bp in length. The 5’-untranslated region (UTR) and 3’-UTR of CmFT were 75 bp and 219 bp in length, respectively. CmFT encoded 174 amino acids, which contained a conserved PBP domain. The homology of FT in Chinese chestnut, Arabidopsis, Cymbidium goeringii, Vitis vinifer, Jatropha curcas, Paeonia suffruticosa, Populus nigra and Cucumis sativus was 87.36%. The results of evolutionary analysis showed that FT homologous genes in different species were highly conservative. Among 13 species, including species above, as well as Glycine max, Camellia oleifera, Lactuca sativa, Brassica napus and Aquilegia coerulea, CmFT of Chinese chestnut had the closest relationship with the FT homologous protein of cucumber, followed by poplar. CmFT was expressed in stem tips, leaves, mixed buds and inflorescences. The expression of CmFT in stem tips and leaves was higher, followed by inflorescences and mixed buds. The expression level of CmFT in leaves gradually increased from April, and reached a peak on July 15th, then rapidly decreased, and increased again on September 5th. The results GFP fluorescence signal observation showed that the green fluorescence in leaf protoplasts of Arabidopsis transformed with 35S::CmFT-GFP recombinant expression vector was only distributed in the nucleus. This result indicated that CmFT protein was located in the nucleus. The results of flowering phenotype analysis showed the number of rosette leaves of 5 transgenic lines, when the flower stalk reached 2-5 cm, was 6.3, 7.2, 6.3, 6.0 and 8.2, which were significantly (p ＜0.05) lower than that of the wild-type Arabidopsis 11.8. This result showed that overexpression of CmFT significantly promoted Arabidopsis flowering. The expression levels of the florigen gene AtFT and its suppressor gene AtTFL1, as well as the photoperiod pathway gene AtSOC1 both in wild-type and transgenic Arabidopsis at 8-leaf stage were higher than those at 4- leaf stage. The expression level of flowering pioneer gene AtLFY changed insignificantly between the two periods. But the expression level of AtLFY in transgenic Arabidopsis leaves at the 8-leaf stage was significantly higher (p ＜0.05) than that in the wild-type plants. In addition, the expression level of the flowering suppressor gene AtFLC in transgenic Arabidopsis was significantly higher (p ＜0.05) than that of the wild type at the 4-leaf stage, but the expression levels between various periods were insignificantly different. The expression level of the photoperiod pathway gene AtCO at 8-leaf stage was significantly (p ＜0.05) lower than that at 4-leaf stage, but there was no significant difference between transgenic and wild- type A. thaliana.【Conclusion】CmFT is a typical member of the PEBP family. The overexpression of CmFT promotes flowering in Arabidopsis. CmFT is the florigen gene of Chinese chestnut.